Int. J. Biol. Sci. 2020, Vol. 16 2063

Ivyspring International Publisher International Journal of Biological Sciences 2020; 16(12): 2063-2071. doi: 10.7150/ijbs.45999 Research Paper KLF10 inhibits cell growth by regulating PTTG1 in multiple myeloma under the regulation of microRNA-106b-5p Mimi Zhou1, Jinqiu Chen2, Hui Zhang2, Hailing Liu2, Huan Yao2, Xiaman Wang2, Wanggang Zhang2, Yingren Zhao1, Nan Yang1

1. Department of Infectious Diseases, the First Affiliated Hospital of Xi’an Jiaotong University, Yanta West Road No. 277, Xi’an 710061, China 2. Department of Hematology, the Second Affiliated Hospital of Xi’an Jiaotong University, West Five Road No. 157, Xi’an 710004, China

 Corresponding author: Nan Yang, Phone number: +8613 572 227 780, E-mail: [email protected].

© The author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/). See http://ivyspring.com/terms for full terms and conditions.

Received: 2020.03.14; Accepted: 2020.05.05; Published: 2020.05.15

Abstract Krüppel-like factor 10 (KLF10) has been identified as an important regulator in carcinogenesis and cancer progression. However, the role of KLF10 in multiply myeloma (MM) development and progression remains unknown. In present study, we found that KLF10 mRNA and were down-regulated in MM tissues and cell lines. Notably, KLF10 inhibited cell proliferation, cell cycle progression and promoted apoptosis in vitro and in vivo. Furthermore, we confirmed that KLF10 inhibited β-catenin nuclear translocation and inhibited PTTG1 transcription. PTTG1 knockdown could mimic the biological effects of KLF10. Moreover, we demonstrated that KLF10 expression was regulated by miR-106b-5p. In MM tissues, miR-106b-5p has an inverse correlation with KLF10 expression. Conclusively, our results demonstrated that KLF10 functions as a tumor suppressor in regulating tumor growth of MM under regulation of miR-106b-5p, supporting its potential therapeutic target for MM.

Key words: KLF10, multiply myeloma, PTTG1, miR-106b-5p, proliferation

Introduction Multiply myeloma (MM) is a plasmablast factors [4]. By binding to Sp-1-GC rich DNA malignancy characterized by heterogenetic plasma sequences, KLF10 regulates transcription and cells (PC) clonal proliferation in the bone marrow [1]. impacts multiple pathways of the physiological and It is the second most common hematological cancer pathological processes, including bone metabolism worldwide and accounts for 20% of all deaths from [5], cardiac hypertrophy [6], neovascularization [7], T hematological malignancy [2]. Despite the survival is cell differentiation [8,9], as well as tumorigenesis. In largely improved in recent year, due to the multiple neoplasm cells, KLF10 was found to be development of the novel chemo-therapies, like deregulated involving proliferation, apoptosis, and proteasome inhibitor and immunomodulator, cell cycle arrest. KLF10 expression in breast combining with autologous stem cell transplant, carcinomas was displayed less than one-half of levels majority of patients become refractory to treatment in normal breast epithelium and highly associated and ultimately relapse, which makes MM remain an with tumor stages [10]. Notably, overexpression of incurable disease [3]. Therefore, the precise molecular KLF10 exerted anti-proliferative effects and induced mechanisms of MM pathogenesis still need to be apoptosis in pancreatic cancer [11,12]. Moreover, elucidated for innovative therapeutic strategies. mRNA expression profiling revealed LSAMP Krüppel-like factor 10 (KLF10), also called TGFβ suppressed osteosarcomas proliferation possibly inducible early gene-1 (TIEG1), is classified as a through upregulation of KLF10 [13]. Therefore, KLF10 member of the Krüppel-like family of transcription has been suggested to be a tumorigenesis suppressor.

http://www.ijbs.com Int. J. Biol. Sci. 2020, Vol. 16 2064

However, the functional role of KLF10 and its reagent (Invitrogen). Total RNA was respectively transcriptional regulation mechanism in MM has not reverse-transcribed using the PrimeScript RT reagent been elucidated yet. kit (Takara, Dalian, China) for mRNA and miRNA In the present study, we demonstrated that First-Strand Synthesis kit (Takara) for miRNA. Real- KLF10 expression was significantly down-regulated time PCR was performed using SYBR Green RT-PCR in human clinical MM and cell lines. We confirmed kit (Takara). was measured in that KLF10 overexpression regulated cell triplicate and data were processed using 2-ΔΔCT proliferation, cycle and apoptosis of MM cells by method and normalized to GAPDH or U6 as the affecting β-catenin signaling pathway. Moreover, we internal control. Primers of KLF10 (HQP018084), demonstrated that KLF10 suppressed PTTG1 PTTG1 (HQP061080), GAPDH (HQP064347), miR- transcription, which plays critical role in MM 106b-5p (HmiRQP0029), and U6 (HmiRQP9001) were development. In addition, we identified KLF10 purchased from GeneCopoeia (Guangzhou, China). expression was regulated by miR-106b-5p in MM cells. Taken together, our findings suggest that KLF10 Protein extraction and Western blot analysis acts as a tumor suppressor in MM progression. Total protein was extracted by RIPA buffer supplemented with protease phosphatase inhibitors Materials and Methods (Roche, Basel, Switzerland). Cytoplasmic and nuclear protein was extracted by the Nuclear and Samples and cell lines Cytoplasmic Extraction kit (Thermo Fisher). Western Primary plasma cells purified by CD138 blot analysis was conducted as described previously immunomagnetic bead (Miltenyi Biotec, Auburn, CA) [14]. from bone marrow were obtained from 38 newly diagnosed MM patients and five healthy donors. Proliferation, apoptosis, and cell cycle assay CD138 flow cytometry determined that plasma cell Cell proliferation rates were evaluated using a purity was routinely ≥90%. This research study was CCK8 assay at 0, 24, 48, and 72 h post-transduction. approved by the Ethical Committee of Second Apoptosis and cell cycle were determined by flow Affiliated Hospital of Xi’an Jiaotong University and cytometry at indicated time points. The detailed all patients provided the informed consents. experiment was performed as previous reported [14]. Human MM cell lines (RPMI8226, NCI-H929, and U266) were purchased from China Center for Luciferase reporter assay Type Culture Collection (Beijing, China) and cultured To illustrate the relationship between KLF10 and in RPMI 1640 media (Gibco, Grand Island, NY, USA) PTTG1, wild-type (wt) (CTCACGCCCGT) or mutant with 10% heat-inactivated foetal bovine serum (mt) promoters of PTTG1 were inserted into pGL3 (Gibco), penicillin (100 U/mL), and streptomycin (100 vectors and co-transfected with plasmids containing μg/mL). HEK293T were grown in DMEM media KLF10 or NC into HEK293T by Lipofectamine 2000 (Gibco) with the same supplement. All cells were Reagent (Thermo Fisher Scientific). Cells were maintained at 5% CO2 and 37 °C temperature in the harvested at 48 h after the transfection and measured incubator. by Dual Luciferase Reporter Assay System (Promega Corporation, Fitchburg, WI, USA). Luciferase Lentivirus transduction and oligonucleotide activities were expressed as the luminescence of transfection Firefly relative to Renilla. To identify the association Lentiviruses encoding KLF10 (LV-KLF10) and between miR-106b-5p and KLF10, HEK293T was control (LV-control) were purchased from Obio co-transfected with wt or mt 3’UTR of KLF10 and (Shanghai, China). RPMI8226 and U266 were infected either miR-106b-5p mimic or miR-control and with lentiviruses at MOI of 100 in the presence of 5 analyzed by the same system. μg/mL Polybrene (Obio). siRNA against PTTG1 Xenograft mouse model (si-PTTG1), non-targeting scrambled siRNA (si-Control), miR-106b-5p inhibitor (anti-miR-106b- Eight 4-6-week-old female BALB/c nude mice 5p), and inhibitor negative control (anti-miR-NC) (Center of Laboratory Animals, Health and Science were synthesized by Sangon Biotech (Shanghai, Center of Xi’an Jiaotong University, Xi’an, China) China). The transfection was performed as described were randomized into two groups. KLF10 or NC before [14]. stably-overexpressed RPMI8226 cells (1×107) were injected subcutaneously with 150 μL Matrigel RNA extraction and quantitative real-time basement membrane matrix (Becton Dickinson) into PCR analysis the mice of each group. Then the diameters were Total RNA was extracted from cells using Trizol measured every 3 days and tumor volume was

http://www.ijbs.com Int. J. Biol. Sci. 2020, Vol. 16 2065

Figure 1. KLF10 is significantly down-regulated in MM tissues and cell lines. (A) Relative KLF10 mRNA expression levels in MM and healthy donors were determined by qRT-PCR. (B) Representative Western blot analysis of KLF10 expression in the MM and healthy donors was shown. (C) KLF10 level was compared between MM tissues of different ISS stage. The expression of KLF10 mRNA (D) and protein (E) in three MM cell lines was significantly decreased compared to that in the nPCs cells. n = three repeats with similar results. *P < 0.05 by ANOVA. **P < 0.01.

calculated as previously described [14]. After 3 weeks, nPCs (Figure 1D-E, respectively, P < 0.05). RPMI8226 the mice were sacrificed by cervical dislocation under and U266 expressing relatively low levels of KLF10 anesthesia with ether and the xenograft tumor tissue were selected for further studies. was fixed in formalin. Animal experiment protocols were approved by the Animal Committee of Xi'an KLF10 promoted apoptosis and inhibits Jiaotong University. proliferation and cell cycle transition in vitro To investigate possible biological function of Statistical analysis KLF10, KLF10-overexpressed MM cell lines Statistical analysis was performed by GraphPad (RPMI8226 and U266) were established by lentivirus Prism 5 software. All data represent the mean ± transduction and confirmed by Western blot (Figure standard deviation (SD), and results were analyzed 2A, P < 0.05). A remarkable increase of the proportion using the Mann–Whitney U test. p < 0.05 was of apoptotic cells was observed in MM cell lines stably considered statistically significant. expressing KLF10 compared with control cells (Figure 2B, P < 0.05). Moreover, upregulation of KLF10 Result triggered significant inhibition of cell proliferation in KLF10 was downregulated in MM primary cells time-dependent manner in both RPMI8226 and U266 (Figure 2C, P < 0.05). In addition, the flow cytometry and cell lines testified that KLF10 overexpression resulted in more We first evaluated the expression of KLF10 in cells arrest in G0/G1 phase and fewer in S phase than bone marrow derived from 38 MM patients and ten NC (Figure 2D, P < 0.05). Compared with health donors. qRT-PCR and Western blot analysis NC-transduced cells, the KLF10-transduced cells exhibited that KLF10 mRNA and protein was expressed less Cyclin D1, Bcl-2, and c- significantly decreased in MM patients compared but more p27 and Bax proteins (Figure 2E, P < 0.05). with health controls (Figure 1A-B, respectively, P < Furthermore, KLF10 knockdown showed opposite 0.05). The GEO dataset (GSE6477) from R2: Genomics effects on NCI-H929 cells (Figure S2). Taken together, Analysis and Visualization Platform (http:// these results indicate that enforced expression of r2.amc.nl) consistently showed down-regulated KLF10 inhibits survival and growth of MM cells. KLF10 in MM (Figure S1, P = 0.0052). Moreover, the expression of KLF10 was associated with ISS stage. KLF10 regulated tumor growth in vivo The data showed that KLF10 was decreased in Finally, we assessed the anti-MM activity of advanced stage (Figure 1C, P < 0.05). We further KLF10 in nude mice bearing subcutaneous RPMI8226 validated the expression of KLF10 in three MM cell xenografts. There was a significant reduction in tumor lines (RPMI8226, NCI-H929, and U266) and normal formation in mice injected with KLF10-overexpressed plasma cells (nPCs). Similarly, the expressions of MM cells when compared with NC (Figure 3A-B, P < KLF10 mRNA and protein were notably 0.05). We also stain the plasma cell marker CD138 downregulated in MM cell lines in comparison with (Figure S3). Additionally, IHC analysis of excised

http://www.ijbs.com Int. J. Biol. Sci. 2020, Vol. 16 2066 tumors confirmed that elevated KLF10 notably expression of Cyclin D1 and c-Myc, which were suppressed the expression of Ki-67 protein, which known as Wnt-downstream targets. Thus, we next indicated the proliferation activity of MM cells (Figure investigated whether Wnt pathway was involved in 3C, P < 0.05). Besides, KLF10-overexpressing tumors KLF10-induced changes in MM. Although the displayed a significantly higher apoptotic index, as cytosolic accumulation remained same, the indicated by the number of TUNEL-positive cells accumulation of β-catenin in the nucleus was largely (Figure 3D, P < 0.05). Together, these results declined in KLF10-transduced cells (Figure 4A, P < demonstrated potent anti–MM activity of KLF10 in 0.05). In addition, the enhanced KLF10 remarkably vivo. repressed the phosphorylation of GSK3β on Ser-9 in comparison with NC (Figure 4B, P < 0.05). Similarly, KLF10 suppressed the activation of Wnt we confirmed that the activation of Wnt signaling was signaling suppressed by KLF10 in subcutaneous RPMI8226 The unrestrained growth of tumor cells is xenografts (Figure S4, P < 0.05). Therefore, we generally attributed to activation of essential demonstrated that Wnt signaling was involved in the pathways. Active Wnt signals are identified as a KLF10-induced anti-MM function. hallmark in MM tumorigenesis [15-17]. In the above results, we found that KLF10 downregulated the

Figure 2. KLF10 inhibits cell proliferation, cell-cycle progression and promotes apoptosis in MM cell. (A) RPMI8226 and U266 cells that were transduced with corresponding KLF10 overexpression vectors were subjected to WB for KLF10. (B) Flow cytometry checked the effects of KLF10 up-regulation on apoptosis. (C) KLF10 overexpression inhibited cell proliferation in RPMI8226 and U266 cells. (D) Effects of KLF10 overexpression on the cell cycle progression of MM cells were measured by flow cytometric analysis. (E) WB measured the cycle- and apoptosis-associated factors. n = six independent experiments. *P<0.05.

http://www.ijbs.com Int. J. Biol. Sci. 2020, Vol. 16 2067

Figure 3. KLF10 inhibits tumor growth and promotes apoptosis in vivo. (A) Representative pictures of MM xenografts from RPMI8226-KLF10 and RPMI8226-control. (B) Tumor growth curve revealed that KLF10 overexpression significantly inhibited tumor growth in vivo. Tumor nodules were subjected to immunohistochemical staining for Ki-67 (C) and TUNEL (D) assays and quantitative analysis. Representative immunostaining and TUNEL assays revealed that KLF10 overexpression significantly decreased the number of Ki-67 positive cells and increased the number of apoptotic cells. *P<0.05.

Figure 4. KLF10 inhibits activation of Wnt signaling. (A) WB showed that KLF10 overexpression suppressed β-catenin nuclear accumulation in RPMI8226 and U266 cells. (B) WB revealed that KLF10 overexpression suppressed GSK3β phosphorylation in MM cells. n = six independent experiments. *P<0.05, **P<0.01.

samples. Both mRNA and protein level of PTTG1 PTTG1 was the direct target of KLF10 in MM were significantly elevated in MM patients compared Well known as a transcription factors, KLF10 with health donors (Figure 5A-B, P<0.05). was involved in the regulation of a variety of in Interestingly, the expression of PTTG1 protein different tissues [18-20]. PTTG1 was a potential target displayed an inverse correlation to KLF10 protein of KLF10 in cardiac hypertrophy [6,21] but their (r=-0.8146, Figure 5C, P < 0.05). The GEO dataset relationship in MM remains unclear. To investigate (GSE6401) from R2: Genomics Analysis and the association between KLF10 and PTTG1, we first Visualization Platform (http://r2.amc.nl) showed an analyzed the expression of PTTG1 in primary MM inverse relationship between KLF10 and PTTG1

http://www.ijbs.com Int. J. Biol. Sci. 2020, Vol. 16 2068

(Figure S5, P = 0.02). We further analyzed the effect of MiR-106b-5p directly downregulated KLF10 in KLF10 on the expression of PTTG1. KLF10 largely MM inhibited the mRNA and protein level of PTTG1 in It is well documented that miRNA comparison with NC (Figure 5D-E, P<0.05). Finally, to translationally represses gene mRNA production by validate KLF10-dependent regulation of PTTG1, we binding to the seed sequence in the 3’-UTR of the cloned the PTTG1 promoter into an expression vector target RNA in various hematological malignancies downstream of the luciferase reporter gene, which [22]. Therefore, we interrogated TargetScan and was co-transfected into HEK293T cells together with miRanda to identify the controlling miRNA of KLF10. KLF10 or NC. Overexpression of KLF10 induced a With conserved target sites and good context score remarkable suppression of the luciferase activity in percentile/mirSVR score, miR-106b-5p was predicted presence of wt promoter but no noticeable changes in as a candidate target in either of two bioinformatics presence of mt one (Figure 5F, P < 0.05). These results databases (Figure 7A). Moreover, miR-106b-5p was demonstrated that KLF10 directly downregulated up-regulated in MM compared to healthy donors PTTG1 through binding to its promoter. (Figure 7B, P < 0.05). To elucidate the association of Inhibition of PTTG1 mimics KLF10-induced miR-106b-5p and KLF10, miR-106b-5p knockdown in biological effects on MM MM cell lines was conducted (Figure 7C, P < 0.05). qRT-PCR and Western blot showed that the To examine the function of PTTG1, we first downregulation of miR-106b-5p promoted the constructed the PTTG1-knockdown MM cells (Figure expression of KLF10 at both mRNA and protein levels 6A, P < 0.05). As expected, the downregulation of (Figure 7D-E, P < 0.05). The luciferase reporter assay PTTG1 promoted the apoptosis, inhibited the demonstrated that miR-106b-5p markedly decreases proliferation, and induced the cycle arrest at G0/G1 the luciferase activity of wt KLF10 3’UTR, but not that phase in MM cells by regulating the related factors of mt KLF10 3’UTR (Figure 7F, P < 0.05). In addition, (Figure 6B-E, P < 0.05). Furthermore, anti-PTTG1 we confirmed an inverse correlation between resulted in β-catenin degradation and the miR-106b-5p and KLF10 expression in MM tissues dephosphorylation of GSK3β (Figure 6F-G, P < 0.05). (Figure 7G, r=-0.7891, P < 0.05). We also found that the Thus, downregulation of PTTG1 could mimic the downregulation of miR-106b-5p could mimic the KLF10-induced the biological effects on MM. KLF10-induced the biological effects on MM (Figure S6, P<0.05). These data strengthen the role of miR-106b-5p as KLF10 negative regulator in MM.

Figure 5. KLF10 inhibited PTTG1 transcription in MM cells. (A) Relative PTTG1 mRNA expression levels in MM and healthy donors were determined by qRT-PCR. (B) Representative Western blot analysis of PTTG1 expression in the MM and healthy donors was shown. (C) A significant inverse correlation between the KLF10 and PTTG1 was observed in MM tissues. KLF10 overexpression inhibited PTTG1 mRNA (D) and protein (E) expression in MM cells. (F) Luciferase reporter assays confirmed that KLF10 inhibited PTTG1 transcription in HEK293 cells. n = three repeats with similar results. *P<0.05, n.s: no significance.

http://www.ijbs.com Int. J. Biol. Sci. 2020, Vol. 16 2069

Figure 6. Inhibition of PTTG1 mimics KLF10-induced biological effects on MM. (A) WB showed the effects of PTTG1 siRNA to knockdown PTTG1. PTTG1 knockdown promoted apoptosis (B) and inhibited cell proliferation (C) and cell cycle progression (D). (E) WB measured the cycle- and apoptosis-associated factors. (F) WB showed that PTTG1 knockdown suppressed β-catenin nuclear accumulation in MM cells. (G) WB revealed that PTTG1 knockdown suppressed GSK3β phosphorylation in MM cells. n = six repeats with similar results. *P<0.05, n.s=no significance.

associated with cell cycle-dependent events and Discussion multiple cell cycle-regulatory molecules [28]. Here, KLF10 encodes a three-zinc-finger Krüppel-like we investigated the activity and mechanism of KLF10 , which is originally cloned from in inhibiting MM cells growth and survival. human osteoblasts (OBs) as a primary response gene In the present study, we found that KLF10 following TGF-β treatment [23]. KLF10 mimics TGF-β expression was nearly absent in MM primary samples action and plays a role in the development of human and cell lines. To address the biological effects of osteosarcoma [13], pancreatic carcinoma [24], breast KLF10 in MM, we demonstrated that KLF10 inhibited cancer [25] and renal cancer [26]. KLF10 deficient mice MM cell proliferation, cell cycle progression and showed more increased cell proliferation in skin and promoted apoptosis by regulating cycle- and earlier onset of skin cancers than control littermates apoptosis- associated factors in vitro. Moreover, we [27]. KLF10 expression was significantly upregulated showed that KLF10 suppressed tumor growth and the by homoharringtonine and Velcade and KLF10 was a Ki67 and TUNEL staining confirmed that KLF10 key regulator which can induce and promote inhibited cell proliferation and promoted apoptosis in apoptosis through the mitochondrial apoptotic vivo. We also confirmed that KLF10 inhibited pathway [23]. KLF10 protein expression was tightly β-catenin nuclear transition and suppressed Wnt

http://www.ijbs.com Int. J. Biol. Sci. 2020, Vol. 16 2070

Figure 7. KLF10 is identified as a direct target of miR-106b-5p in MM. (A) miR-106b-5p and its putative binding sequence in the 3’-UTR of KLF10. The mutant binding site was generated in the complementary site for the seed region of miR-106b-5p. (B) miR-106b-5p was up-regulated in MM tissues compared to healthy donors. (C) miR-106-5p was knockdown by the miR-106b-5p inhibitors and measured by qRT-PCR. (D) qRT-PCR analysis of KLF10 mRNA expression in MM cells with anti-miR-106b-5p or anti-miR-NC vector transfection. (E) miR-106-5p knockdown increased the expression of KLF10 protein in MM cells. (F) miR-106b-5p significantly suppresses the luciferase activity that carried wild-type (wt) but not mutant (mt) 3'-UTR of KLF10. (G) A significant inverse correlation between the mRNA levels of KLF10 and miR-106b-5p was observed in MM tissues. n = six repeats with similar results. *P<0.05, n.s=no significance.

pathway. KLF10 inhibited PTTG1 expression by an attractive therapeutic target for MM treatment. regulating its transcription. PTTG1 knockdown mimics the biological effects of KLF10 in vitro. Supplementary Material Increasing evidence confirmed that abnormal Supplementary figures and tables. miRNAs have been verified to be crucial regulators in http://www.ijbs.com/v16p2063s1.pdf the initiation and progression of human cancer. We searched the bioinformation database and showed Acknowledgements that miR-106b-5p could bind to KLF10 3’UTR. This research was supported by Institutional Luciferase reporter assays showed miR-106b-5p could Foundation of the First Affiliated Hospital of Xi’an directly bind to the 3’UTR of KLF10 and regulated Jiaotong University (2019QN-24) and Natural Science KLF10 expression. Moreover, miR-106b-5p negatively Basic Research Program of Shaanxi (2020JQ-498). regulated the expression of KLF10 mRNA and protein in MM cells. In MM tissues, miR-106b-5p showed an Authors' contributions inverse correlation with KLF10 expression. These data MMZ and NY conceived and designed the further confirm that KLF10 down-regulation was experiments; MMZ, JQC, HZ, HLL, HY, and XMW caused by miR-106b-5p overexpression. Conclusion, performed the experiments; NY, WGZ and YRZ these results suggest that KLF10 was a downstream analyzed the data; MMZ, JQC and HZ contributed target of miR-106b-5p in MM. reagents/materials/analysis tools; NY wrote the We demonstrated that KLF10 was down- paper. All authors read and approved the final regulated in MM tissues and cell lines. We confirm manuscript. that KLF10 inhibits cell proliferation, cell cycle progression and promotes apoptosis in vitro and in Competing Interests vivo. KLF10 inhibited β-catenin nuclear translocation. The authors have declared that no competing KLF10 inhibited PTTG1 expression by regulating its interest exists. transcription. Additionally, we determined that miR- 106b-5p regulated KLF10 expression in MM cells. These findings support the potential role of KLF10 as

http://www.ijbs.com Int. J. Biol. Sci. 2020, Vol. 16 2071

25. Hsu CF, Sui CL, Wu WC, Wang JJ, Yang DH, Chen YC, et al. Klf10 induces cell References apoptosis through modulation of BI-1 expression and Ca2+ homeostasis in 1. Palumbo A, Anderson K. Multiple myeloma. The New England journal of estrogen-responding adenocarcinoma cells. The international journal of medicine. 2011; 364: 1046-60. biochemistry & cell biology. 2011; 43: 666-73. 2. Zweegman S, Palumbo A, Bringhen S, Sonneveld P. Age and aging in blood 26. Ivanov SV, Ivanova AV, Salnikow K, Timofeeva O, Subramaniam M, Lerman disorders: multiple myeloma. Haematologica. 2014; 99: 1133-7. MI. Two novel VHL targets, TGFBI (BIGH3) and its transactivator KLF10, are 3. Mahindra A, Hideshima T, Anderson KC. Multiple myeloma: biology of the up-regulated in renal clear cell carcinoma and other tumors. Biochemical and disease. Blood reviews. 2010; 24 Suppl 1: S5-11. biophysical research communications. 2008; 370: 536-40. 4. Subramaniam M, Hawse JR, Rajamannan NM, Ingle JN, Spelsberg TC. 27. Song KD, Kim DJ, Lee JE, Yun CH, Lee WK. KLF10, transforming growth Functional role of KLF10 in multiple disease processes. BioFactors. 2010; 36: factor-beta-inducible early gene 1, acts as a tumor suppressor. Biochemical 8-18. and biophysical research communications. 2012; 419: 388-94. 5. Subramaniam M, Gorny G, Johnsen SA, Monroe DG, Evans GL, Fraser DG, et 28. Dhyani A, Machado-Neto JA, Favaro P, Saad ST. ANKHD1 represses p21 al. TIEG1 null mouse-derived osteoblasts are defective in mineralization and (WAF1/CIP1) promoter and promotes multiple myeloma cell growth. in support of osteoclast differentiation in vitro. Molecular and cellular biology. European journal of cancer. 2015; 51: 252-9. 2005; 25: 1191-9. 6. Rajamannan NM, Subramaniam M, Abraham TP, Vasile VC, Ackerman MJ, Monroe DG, et al. TGFbeta inducible early gene-1 (TIEG1) and cardiac hypertrophy: Discovery and characterization of a novel signaling pathway. Journal of cellular biochemistry. 2007; 100: 315-25. 7. Wara AK, Foo S, Croce K, Sun X, Icli B, Tesmenitsky Y, et al. TGF-beta1 signaling and Kruppel-like factor 10 regulate bone marrow-derived proangiogenic cell differentiation, function, and neovascularization. Blood. 2011; 118: 6450-60. 8. Cao Z, Wara AK, Icli B, Sun X, Packard RR, Esen F, et al. Kruppel-like factor KLF10 targets transforming growth factor-beta1 to regulate CD4(+)CD25(-) T cells and T regulatory cells. The Journal of biological chemistry. 2009; 284: 24914-24. 9. Venuprasad K, Huang H, Harada Y, Elly C, Subramaniam M, Spelsberg T, et al. The E3 ubiquitin ligase Itch regulates expression of transcription factor Foxp3 and airway inflammation by enhancing the function of transcription factor TIEG1. Nature immunology. 2008; 9: 245-53. 10. Subramaniam M, Hefferan TE, Tau K, Peus D, Pittelkow M, Jalal S, et al. Tissue, cell type, and breast cancer stage-specific expression of a TGF-beta inducible early transcription factor gene. Journal of cellular biochemistry. 1998; 68: 226-36. 11. Tachibana I, Imoto M, Adjei PN, Gores GJ, Subramaniam M, Spelsberg TC, et al. Overexpression of the TGFbeta-regulated encoding gene, TIEG, induces apoptosis in pancreatic epithelial cells. The Journal of clinical investigation. 1997; 99: 2365-74. 12. Jiang L, Chen Y, Chan CY, Wang X, Lin L, He ML, et al. Down-regulation of stathmin is required for TGF-beta inducible early gene 1 induced growth inhibition of pancreatic cancer cells. Cancer letters. 2009; 274: 101-8. 13. Baroy T, Kresse SH, Skarn M, Stabell M, Castro R, Lauvrak S, et al. Reexpression of LSAMP inhibits tumor growth in a preclinical osteosarcoma model. Molecular cancer. 2014; 13: 93. 14. Yang N, Chen J, Zhang H, Wang X, Yao H, Peng Y, et al. LncRNA OIP5-AS1 loss-induced microRNA-410 accumulation regulates cell proliferation and apoptosis by targeting KLF10 via activating PTEN/PI3K/AKT pathway in multiple myeloma. Cell death & disease. 2017; 8: e2975. 15. van Andel H, Kocemba KA, de Haan-Kramer A, Mellink CH, Piwowar M, Broijl A, et al. Loss of CYLD expression unleashes Wnt signaling in multiple myeloma and is associated with aggressive disease. Oncogene. 2017; 36: 2105-15. 16. Zhao JJ, Lin J, Zhu D, Wang X, Brooks D, Chen M, et al. miR-30-5p functions as a tumor suppressor and novel therapeutic tool by targeting the oncogenic Wnt/beta-catenin/BCL9 pathway. Cancer research. 2014; 74: 1801-13. 17. Bjorklund CC, Ma W, Wang ZQ, Davis RE, Kuhn DJ, Kornblau SM, et al. Evidence of a role for activation of Wnt/beta-catenin signaling in the resistance of plasma cells to lenalidomide. The Journal of biological chemistry. 2011; 286: 11009-20. 18. Chen GG, Xu H, Lee JF, Subramaniam M, Leung KL, Wang SH, et al. 15-hydroxy-eicosatetraenoic acid arrests growth of colorectal cancer cells via a peroxisome proliferator-activated gamma-dependent pathway. International journal of cancer. 2003; 107: 837-43. 19. Jiang L, Lai YK, Zhang JF, Chan CY, Lu G, Lin MC, et al. Transactivation of the TIEG1 confers growth inhibition of transforming growth factor-beta-susceptible hepatocellular carcinoma cells. World journal of gastroenterology. 2012; 18: 2035-42. 20. Wu MJ, Wu WC, Chang HW, Lai YT, Lin CH, Yu WC, et al. KLF10 affects pancreatic function via the SEI-1/p21Cip1 pathway. The international journal of biochemistry & cell biology. 2015; 60: 53-9. 21. Bos JM, Subramaniam M, Hawse JR, Christiaans I, Rajamannan NM, Maleszewski JJ, et al. TGFbeta-inducible early gene-1 (TIEG1) mutations in hypertrophic cardiomyopathy. Journal of cellular biochemistry. 2012; 113: 1896-903. 22. Liu Z, Wang Y, Dou C, Sun L, Li Q, Wang L, et al. MicroRNA-1468 promotes tumor progression by activating PPAR-gamma-mediated AKT signaling in human hepatocellular carcinoma. Journal of experimental & clinical cancer research : CR. 2018; 37: 49. 23. Jin W, Di G, Li J, Chen Y, Li W, Wu J, et al. TIEG1 induces apoptosis through mitochondrial apoptotic pathway and promotes apoptosis induced by homoharringtonine and velcade. FEBS letters. 2007; 581: 3826-32. 24. Chang VH, Chu PY, Peng SL, Mao TL, Shan YS, Hsu CF, et al. Kruppel-like factor 10 expression as a prognostic indicator for pancreatic adenocarcinoma. The American journal of pathology. 2012; 181: 423-30.

http://www.ijbs.com